At present the properties enabling Escherichia coli to act as one of the major opportunistic pathogens are not well understood. The E. coli cytolysin/hemolysin (hly) plays a contributory role in the virulence of extraintestinal disease, however, the nature of the interaction of the hly with host cells on a molecular levsel is unknown. The Hly polypeptide subunit is 110,000 daltons in molecular mass and its predicted amino acid sequence reveals domain-like elements based on hydropathy and isoelectric point characteristics. An objective of this project will be to delineate Hly-host cell interaction by use of genetically engineered forms of the hly protein, synthetic peptides, chemical cleavage fragments of the hly protein, and both specific polyclonal and monoclonal antibodies. We will develop the domain-specific reagents for use in in vitro and in vivo assays of the activities associated with the hemolysin. This type of analysis has been productively utilized in studies of diphtheria toxin and bacterial fimbriae and will enable an understanding of an important cytolysin. The second main objective of this project is the continued study of the function and regulation of transcription and translation of the four cistrons responsible for the E. coli hly. We will assay the altered function and cellular location of in vitro constructed mutations of each of the four cistrons. These will be made by either insertion of synthetic oligonucleotides or site-specific oligonucleotide-directed mutagenesis. We will quantitate and map the mRNAs by S-1 nuclease protection, Northern blotting and primer extension techniques. These genetic and physical studies will lead to an understanding of the factors affecting the expression of this virulence factor. Overall, the long term goal of this research program is to identify and characterize the genetic and physiological nature of bacterial factors influencing E. coli extraintestinal infections. This type of information is of great value in the development of selective strategies for the prevention and treatment of serious bacterial diseases.